Journal: Cell Communication and Signaling : CCS

Article Title: Epstein–Barr virus tegument protein BGLF2 in exosomes released from virus-producing cells facilitates de novo infection

doi: 10.1186/s12964-022-00902-7

Figure Lengend Snippet: Exosomal BGLF2 enhances the infectivity of EBV. A and B Akata(-) cells were infected with wildtype ( A ) and BGLF2-KO EBV ( B ) in the presence of BGLF2-containing exosomes. After 2 days, GFP positivity was determined by FACS. Results are presented as the mean ± SE of at least three independent experiments and as the relative infectivity to control treatment with PBS (infectivity value of 1). Double asterisks, p < 0.01; n.s., not significant. C The workflow of sample collection for RNA-seq/qRT-PCR analyses. Akata(-) cells were pretreated with exosomes for 16 h, and then infected with BGLF2-KO EBV. The infected cells were collected via FACS at 24 hpi. Total RNA extracted from the sorted cells was subjected to RNA-seq and qRT-PCR. D Read counts of EBV genes in BGLF2-KO EBV-infected cells that were treated with BGLF2-containing or control exosomes by RNA-seq. E Gene expression levels were normalized as counts per million followed by log 2 -transformation with a pseudo-count of 1. Each bar indicates the log 2 fold change of a viral gene expression between treatment with exosomes with or without BGLF2. Viral gene expression kinetics are categorized into five groups: latent, immediate early, early, leaky late, and late . F Validation of enhanced EBV gene expression by qPCR. Data are presented as the mean ± SE. Samples were tested in duplicate. Asterisk, p < 0.05; double asterisks, p < 0.01; n.s., not significant. G DAVID analysis of RNA-seq data from BGLF2-KO EBV-positive cells treated with BGLF2-containing or control exosomes. FDR, false discovery rate. H HEK293 cells were treated with BGLF2-containing exosomes for 16 h. Cells were challenged with transfection of poly(I:C) (2 μg/ml) for 2.5 h. RT-qPCR was conducted. Data are presented as the mean ± SE of three independent experiments. Asterisk, p < 0.05; n.s., not significant. H Exosomal BGLF2 inhibited type I IFN signaling. AGS/EBV-EGFP cells were pretreated with exosomes for 16 h, and then infected with or without IFN alpha (1000 Unit/mL) for 1 h. Lysates were analyzed by immunoblotting using the indicated antibodies. BGLF2-HA was detected using the anti-HA antibody. J Type I IFN inhibited EBV infection. Akata(-) cells were infected with EBV-EGFP in the presence of IFN alpha and beta (100 or 500 Unit/mL). After 2 days, GFP positivity was determined by FACS. Results are presented as the mean ± SE of three independent experiments. Double asterisks, p < 0.01; n.s., not significant

Article Snippet: Recombinant human type I IFN alpha (Cat# 11200-1) and IFN beta (Cat# 11415-1) were purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Infection, RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Transformation Assay, Transfection, Western Blot

Journal: bioRxiv

Article Title: Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

doi: 10.1101/2021.11.23.469755

Figure Lengend Snippet: pDCs were either mock treated or exposed to the SARS-CoV-2 FR2020 early Wuhan-like strain or the SARS-CoV-2 alpha variant B.1.1.7 (0.1 MOI). Supernatants were collected at indicated time points and the production of type I IFNα (A) and CXCL10 (B) was quantified. The FR2020 strain was used in subsequent experiments where pDC were either mock treated (mock, grey), exposed to SARS-CoV-2 at 1 MOI (SARS-2, purple), TLR7 (2.5 μg/mL R837, blue) or TLR3 agonist (800 ng/mL poly(I:C), pink). Supernatants were collected after 24 hrs and analyzed for type I IFNα (C), IFNβ (D), type II IFNγ (E), type III IFNλ1 (F), IL-6 (G), IL-8 (H), CXCL10 (I) and TNFα (J) expression by ELISA. To evaluate the cytokine response to viral titers and exposure duration, pDCs were exposed to increasing viral inoculums (MOI of 0.01, 0.1 and 1) and IFNα2a mRNA expression was quantified at 24 hrs (K) and IFNα protein secretion at 24, 48, 72 and 96 hrs (L). Graph depicting simple linear regression of IFNα protein with time of exposure (M). Bars and lines represent mean values and symbols represent individual pDC donors (n=3-4). Equal symbols represent equal donors (A-B and C-L). Statistical significance was determined using the ratio paired student T test and compared the treated condition with the time point-matched mock condition (A-L) and simple linear regression (M). *

Article Snippet: To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.

Techniques: Variant Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

doi: 10.1101/2021.11.23.469755

Figure Lengend Snippet: pDCs were isolated from PBMCs using negative selection and either mock treated (mock) or exposed to SARS-CoV-2 (1 MOI, SARS-2). Supernatants were collected at indicated time points and the production of type I IFNα (A), type III IFNλ1 (B), IL-6 (C), IL-8 (D), CXCL10 (E) and TNFα (F) was quantified by ELISA. Bars and lines represent mean values and symbols represent individual donors (n=3). Statistical significance was determined using the ratio paired student T test by comparing virus exposed cell culture supernatant to the mock control of the matched time point. *

Article Snippet: To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.

Techniques: Isolation, Selection, Enzyme-linked Immunosorbent Assay, Virus, Cell Culture, Control

Journal: bioRxiv

Article Title: Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

doi: 10.1101/2021.11.23.469755

Figure Lengend Snippet: To assess whether pDCs could mount protection against SARS-CoV-2, conditioned medium from SARS-CoV-2-exposed pDC cultures (d3 post inoculation with 1 MOI) was added to A549 hACE2 lung epithelial cells (A) or Calu-3 (B) cultures followed by SARS-CoV-2 inoculation. The cell cultures were conditioned with normal medium ( -, grey), pDC supernatant (pDC mock, blue) or SARS-CoV-2-inoculated pDC supernatant (pDC SARS-2, purple), prior to infection with SARS-CoV-2 (0.1 MOI). Supernatants were collected and viral outgrowth was determined 48h post infection. To investigate a potential dose-response, SARS-CoV-2-inoculated pDC supernatant was 3-fold serially diluted prior to addition to Calu-3 cells (purple-pink gradient, B). To determine the involvement of type I IFNs, Calu-3 cells and SARS-CoV-2-inoculated pDC supernatants were pre-treated with antibodies blocking the type I IFN receptor and antibodies neutralizing type I IFNα (IFN-I block) or isotype control antibodies (isot ctrl), prior to the addition of conditioned medium to the cells and infection (C). All antibodies were used at a final concentration of 10 μg/mL. Bars and lines represent median values and symbols represent individual HSPC-pDC donors (n=3-5) Equal symbols represent equal donors. Statistical significance was determined using the ratio paired student T test. *

Article Snippet: To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.

Techniques: Infection, Blocking Assay, Control, Concentration Assay

Journal: bioRxiv

Article Title: Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

doi: 10.1101/2021.11.23.469755

Figure Lengend Snippet: Using CRISPR/Cas9, MyD88 knock-out (KO) and AAVS1 KO (control) pDCs were generated. MyD88 protein levels in KO and control pDCs were analyzed by western blotting (A) and cellular DNA was sequenced to perform an Inference of CRISPR Edits (ICE) analysis (B). MyD88 KO and control pDCs were either mock treated (mock) or exposed to SARS-CoV-2 (SARS-2, 1 MOI), supernatants were collected at indicated time points and analyzed for CXCL10 (C) and type I IFNα (D). Type I IFNα production was then determined in cell culture supernatant from SARS-CoV-2 exposed TRIF KO or TRIG+MyD88 KO (E) and RIG-I KO or RIG-I+MyD88 KO (F) pDCs. Bars represent mean values and equal symbols represent equal donors (n=2).

Article Snippet: To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.

Techniques: CRISPR, Knock-Out, Control, Generated, Western Blot, Cell Culture

Journal: bioRxiv

Article Title: Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

doi: 10.1101/2021.11.23.469755

Figure Lengend Snippet: Using CRISPR/Cas9, TLR7 knock-out (KO) and AAVS1 KO (control) pDCs were generated and cellular DNA was sequenced for ICE analysis (A). For functional evaluation each KO pDC donor was stimulated with TLR3 (800 ng/mL poly(I:C, pink) or TLR7 (2.5 μg/mL R837, blue) agonist; supernatant was collected after 24 hrs and analyzed for IFNα (B) and CXCL10 (C) protein expression by ELISA. AAVS1 KO and TLR7 KO pDCs were either mock treated (mock) or exposed to SARS-CoV-2 (SARS-2, 1 MOI), supernatants were collected at indicated time points and analyzed for type I IFNα (D) and CXCL10 (E) proteins. Wild type pDCs were exposed to SARS-CoV-2 (0.5 MOI) in the absence or presence of an IRAK4 inhibitor (10 μM), 24 hrs after virus exposure the cell culture supernatants were analyzed for production of type I IFNα, CXCL10 and IL-6 proteins (F). IL-6 protein quantification in AAVS1 KO and TLR7 KO pDCs after SARS-CoV-2 exposure (1 MOI) at indicated time points (G). Bars represent mean values and equal symbols represent the donors used throughout the experiments (n=4). Statistical significance was determined using the ratio paired student T test for agonist or virus treated cells and compared to their respective mock treated conditions, or by unpaired T test when comparing matched conditions between different KOs. *

Article Snippet: To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.

Techniques: CRISPR, Knock-Out, Control, Generated, Functional Assay, Expressing, Enzyme-linked Immunosorbent Assay, Virus, Cell Culture

Journal: bioRxiv

Article Title: Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

doi: 10.1101/2021.11.23.469755

Figure Lengend Snippet: Using CRISPR/Cas9, TLR2 KO and AAVS1 KO (control) pDCs were generated, cellular DNA was sequenced for ICE analysis (A) and cells were evaluated functionally by exposure to two different TLR2 agonists; Pam2CSK4 (5 ng/mL, yellow) and Pam3CSK4 (50 ng/mL, orange) (B). Subsequently, AAVS1 KO and TLR2 KO pDCs were either mock treated (mock, grey), exposed to SARS-CoV-2 (SARS-2, 0.5 MOI, purple) or TLR7/8 agonist (2.5 μg/mL R848, red) and supernatants were collected after 24 hrs to quantify type I IFNα (C) and IL-6 (D) protein concentrations. To investigate if pDCs sensed the spike or envelope SARS-CoV-2 proteins, AAVS1 KO and TLR2 KO pDCs were exposed to SARS-CoV-2 (SARS-2, 0.5 MOI, purple) recombinant SARS-CoV-2 spike (S, 1 μg/mL, dark green) or envelope (E, 1 μg/mL, light green) proteins and IL-6 (E) and type I IFNα (F) protein concentrations were quantified. Peripheral blood pDCs were isolated from PBMCs by negative selection and exposed to SARS-CoV-2 (1 MOI, purple), TLR7 agonist (2.5 μg/mL R837, blue) agonist, or E protein (1 μg/mL, light green) for 24 hrs and the concentration of IL-6 (G) and type I IFNα (H) was quantified in cell culture supernatants by ELISA. Bars represent mean values and equal symbols represent equal donors (n=4). Statistical significance was determined using the ratio paired student T test for agonist or virus treated cells and compared to the mock treated condition, or by unpaired T test when comparing matched conditions between different KOs. *

Article Snippet: To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.

Techniques: CRISPR, Control, Generated, Recombinant, Isolation, Selection, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Virus

Journal: bioRxiv

Article Title: Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

doi: 10.1101/2021.11.23.469755

Figure Lengend Snippet: The effect of CD304 signaling on IFNα production was tested by incubating pDCs and peripheral blood isolated pDCs (blood pDC) with anti-CD304 antibody (aCD304) or isotype control antibody (isot ctrl) (10 μg/mL) for 15 minutes prior to stimulation with TLR7 agonist (2.5 μg/mL R837). Cell culture supernatants were harvested after 24 hrs to quantify type I IFNα by ELISA (A). Inference of CRISPR Edits (ICE) analysis of CD304 KO pDCs (B). AAVS1 KO and CD304 KO pDCs were either mock treated (mock) or exposed to SARS-CoV-2 (1 MOI), cell culture supernatants were collected at indicated times and analyzed for type I IFNα (C) IL-6 (D) and CXCL10 (E) protein concentrations. Bars and red lines represent mean values, equal symbols represent equal donors (n=4-5). Statistical significance was determined using the ratio paired student T test for agonist or virus treated cells and compared to the time point-matched mock treated condition, or by unpaired T test when comparing matched conditions between different KOs. *

Article Snippet: To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.

Techniques: Isolation, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, CRISPR, Virus